Product Description

The ISOLATE II RNA/DNA/Protein Kit provides a simple, efficient column-based method for the sequential isolation of total RNA, genomic DNA and proteins from a wide variety of starting materials, without the need for hazardous reagents such as phenol.

By combining the stringency of guanidine-thiocyanate lysis with the speed and ease-of-use of three silica-membrane purification columns, the ISOLATE II RNA/DNA/Protein Kit provides a fast method for the purification of high-quality total RNA, genomic DNA and proteins from a single sample of cultured cells, mammalian tissue, biofluids (including saliva, urine, semen and blood), bacteria, yeast, fungi or plants.

For applications that are sensitive to very small amounts of DNA, residual amounts of DNA remaining can be removed using a convenient on-column DNase treatment with RNase-free DNase I that is supplied with the kit.

The ISOLATE II RNA/DNA/Protein Kit allows for highly reliable integrative analysis of the transcriptome, genome and proteome, since the RNA, DNA and proteins are derived from the same sample and the same cell population.

The purified nucleic acids and proteins are of the highest purity and integrity and the ISOLATE II RNA/DNA/Protein Kit has been designed to deliver optimal performance in a wide variety of downstream applications including miRNA profiling using the EPIK Panel and Select Assays, novel biomarker discovery, siRNA gene silencing studies, gene expression profiling, as well as epigenetics, genotyping, transgenic analysis and cell line characterization.

Applications

miRNA profiling

RT-qPCR

End-point RT-PCR

Southern/northern/western, dot and slot blotting

NGS DNA and RNA-Seq

Genotyping

Restriction digestion

2D gel electrophoresis

Mass spectroscopy

Bisulfite conversion/methylation analysis

High quality total RNA isolation

ISOLATE II RNA/DNA/Protein Kit and a similar kit from Supplier Q was used to isolate total RNA from liver tissues. Total RNA purified from both kits had a RIN>9 (Agilent Bioanalyser 2100), however the ISOLATE II RNA/DNA/Protein Kit also purified increased amounts of small RNA with no further processing required and without use of phenol for unbiased recovery.

Isolation of total RNA from a wide variety of samples

Total RNA was isolated from 105 HeLa cells (lanes 1-2), 3 x 105 yeast cells (lanes 3-4) and 3 x 107E. coli cells (lanes 5-6) using ISOLATE II RNA/DNA/Protein Kit and run on a MOPS agarose gel. The results illustrate that all of the RNA was recovered from all three sample types, including the small RNA fraction.

Fast purification of high-quality total RNA, genomic DNA and proteins from a single sample of cultured cells, mammalian tissue, biofluids, bacteria, yeast, fungi or plants.

Shipping conditions

FAQs

The columns supplied with the ISOLATE II kits may appear similar, but each type has been optimized to work within the buffer system supplied with the corresponding kit. The swapping of columns (or buffers) between kits may lead to no recovery of nucleic acid whatsoever, or at the very least severely impaired purification.

Interruption of the DNA/RNA extraction process is possible after the sample lysis only. It is possible to homogenize and lyse the samples and to store them in the freezer until use for RNA extraction. RNA clean up with a column cannot be interrupted and we recommend to avoid delays during the column purification process. If a delay is unavoidable the columns should be stored on ice.

An increase of A230 values may be caused by different substances, like carbohydrates, peptides and phenol. A bad A230/A260 ratio in RNA samples is mostly due to a contamination with guanidinium thiocyanate which is present in several reagents used for RNA extraction, for instance in the lysis buffer. In contrast to a bad A280/A260 ratio it does not automatically reflect a bad RNA quality. Currently there is no consensus about a lower limit of this ratio and mostly a carry-over of guanidinium thiocyanate does not affect the reliability of downstream applications. Nevertheless, an extra washing step with RW2 would be helpful to avoid this problem. And it would be helpful to pipet the flow-through out of the collection tube, instead of pouring it off.

The ISOLATE II DNA/RNA/Protein Kit recovers all kinds of protein, therefore it is also suitable for extraction and examination of phosphoproteins. Phosphatase inhibitors are not included within the kit and may be added to the elution or lysis buffer to prevent degradation.

Several possibilities exist. If you are using our column based extraction kit like the ISOLATE II RNA Mini kit it would be necessary to decrease the sample amount of such samples or to increase the amount of the lysis buffer RLY to prevent clogging of the columns. Another possibility would be a pre-extraction with TRIsure (BIO-38032) and to clean up the RNA containing aqueous phase with the column based ISOLATE II RNA kits. You should follow the TRIsure protocol up to the phase separation step. Then mix the aqueous phase with a volume of ethanol and load it onto the column. From there you should proceed with the regular ISOLATE II RNA Mini kit protocol.

Agarose gel analysis of RNA samples can be both valuable and misleading. The pattern of bands on the gel can not only be indicative of the quality of the sample, but equally it can also only indicate that the gel tank, buffer or agarose is contaminated with RNase. A safer measure is to use a Bioanalyzer and look at the RIN value (RNA Integrity Number), which should be as close to 10 as possible, indicating that the RNA is not degraded. A spectrophotometer (ideally a microfluidic one) can also be used to determine the ratio of A260 to A280 for purity determination.

All these methods give an idea of the quality of total RNA which tends to be dominated with rRNA and tRNA. It is not uncommon for an agarose gel to show an abundance of 18S and 23S rRNA but on analysis for there to be little of the transcript of interest. Ideally RNA quality control should include an assessment of the presence of common transcripts (from reference genes, such as GAPDH) using RT-PCR or RT-qPCR.

If the yield of RNA is low, it is best to first check that all the solutions used and the equipment employed is largely free of RNase. Solutions should be prepared with the highest quality reagents available, preferably ones that have a certificate of analysis to show that they are free of DNase and RNase. If there is confidence that RNase contamination is at a minimum, then it may be worth ensuring that the sample is properly homogenised before lysis and to check a sample of the lysed material under a light microscope to ensure that all the cells are disrupted.

RNA samples that have been contaminated with RNase (either during processing or those that have been sent from another lab) can be purified using the ISOLATE II RNA Micro Kit.

For the optimal recovery of large RNA/DNA fragments it is necessary to optimize the elution step. To increase the recovery rate it would be helpful to use a larger volume for elution, incubate the column with the elution buffer prior to centrifugation and use repeated elution steps. To avoid excessive dilution it may be helpful to reapply the eluted fraction again. Furthermore it could be helpful for DNA elution to heat the elution buffer to 70°C.

Reviews

"I found the ISOLATE II RNA/DNA/Protein Kit very easy to use, the sample preparat ..."

"This kit (ISOLATE II RNA/DNA/Protein) means we would require less plates of cells, less steps in dividing up a sample (such as tissue) and most importantly time in sample prep."

Michael Epis, The Harry Perkins Institute of Medical Research

I found the ISOLATE II RNA/DNA/Protein Kit very easy to use, the sample preparation was quick, efficient and a more streamlined protocol compared to other commercially available kits, yielding high quality products with superior separation.